Apoptosis is a fundamental biological mechanism of the cell, by which it executes its own death upon receiving appropriate signals. Our long-term objective is to understand the molecular mechanisms of this process to develop effective therapeutic strategies for a number of pathological conditions including cancer, neurodegenerative diseases or aging, where apoptosis plays a key role. Death signals appear to impinge on mitochondria in many cases, resulting in the release of key effector proteins from the intermembrane space. We have shown that Bcl-2 family proteins permeabilize the outer membrane directly using in vitro systems such as outer membrane vesicles and cardiolipin containing liposomes. However, cardiolipin appears not to be abundant in the outer membrane of mitochondria. Therefore, we hypothesize that components in the outer membrane are required for Bcl-2 family proteins to permeabilize it. We will propose a biochemical approach to test this hypothesis. The specific aims are to: 1. Investigate which components, either lipids or proteins, or both, in the outer membrane are required for permeabilization. We will examine this by loading detergent extracted components from isolated outer membrane vesicles into protein-free lipid vesicles. We will treat extracted components of the outer membrane with proteases and reconstitute them into liposomes to see permeabilization of these proteo-liposomes is compromised. If so, it is likely that proteins are responsible for permeabilization induced by Bax/Bid. 2. Fractionate and identify factor(s) that is co-operating with Bax/Bid or Bid alone to permeabilize the outer membrane. We will fractionate detergent extracted outer membrane proteins to identify the factor(s) that confer the activity to liposomes when reconstituted. We will also test if a membrane incorporated proapoptotic multi-domain Bcl-2 family member, such as Bax or Bak, is sufficient for Bid to permeabilize the membrane by incorporating them into liposomes with low levels of cardiolipin.